Exploitation of the mucosal immune system offers several advantages from a vaccine point of view. Mucosal vaccines may achieve both systemic and local mucosal immune protection against infectious micro-organisms of which many gain access to the body via mucosal membranes. There is a growing interest for oral vaccines and for the possibility of using such vaccines to protect against infectious diseases affecting not only mucosal surfaces but also against diseases like HIV, polio etc.
Mucosally active vaccines containing recombinant protein antigens would have many immunological and economic advantages for inducing protective immunity against a wide variety of mucosal and systemic pathogens.
Recent results show that antigens incorporated into iscoms are highly immunogenic by mucosal routes, inducing strong T cell mediated immune responses that include secretory IgA antibodies, Th1 dependent delayed type hypersensitivity (DTH) and cytokine production, as well as very strong class I MHC restricted CD8+ T cell responses. Serum antibody production is also primed, but this is relatively less efficient (3, 11).
The particulate nature of iscoms allows them to preferentially target and activate accessory cells such as macrophages and dendritic cells (DC)(14-17). Iscoms induce normal responses in IL4KO, but not in IL12KO mice (3, 12 and 18). In addition, iscoms stimulate the production of mediators such as IL1, IL6 and IL12 from macrophages and DC and in vivo depletion of macrophages markedly reduces the adjuvant effects of iscoms (15, 18-22).
There is a group of bacterial toxins that exert strong enzymatic activity on mammalian cells, such as E. coli heat-labile toxin (LT) and choler toxin (CT). They act by ADP-ribosylation of GTP-binding proteins in the cell membrane of the target cells, resulting eventually in the formation of large quantities of intracellular cAMP. The increase in cAMP may then act to immunomodulate many diverse immune reactions such as increasing B lymphocyte differentiation, augmenting co-stimulation of antigen-presenting cells, inhibiting or promoting various T cell functions or modulating apoptotis in lymphoid cells. They are therefore potent adjuvants.
CT is composed of five enzymatically inactive, non-toxic B-subunits (CTB) held together in a pentamere structure surrounding a single A-subunit that contains a linker to the pentamere via the A2 fragment (CTA2) and the toxic enzymatically active A1-fragment (CTA1) of the molecule.
The toxic CTA1 has strong ADP-ribosyl transferase activity. This results in activation of adenylate cyclase and the subsequent intracellular increase in cAMP.
CTB binds to the ganglioside GM1-receptor, present on most mammalian cells including lymphocytes and gut epithelial cells. CTB has been integrated in ISCOMS as a mucosa targeting molecule EP 97905539.9 and also together with antigens that do not easily penetrate mucosas EP 97905541.5, in order to direct orally administrated iscoms to the mucosa.
Although it has been shown that CT is a potent inducer of most T cell dependent responses when given orally (3, 4), it has also been reported that it may be less efficient at stimulating CD4+ Th1 cells than Th2 cells (5-7). Furthermore, it is not widely accepted as being able to prime CD8+ T cells, while the toxicity of intact CT is likely to prevent its use as a practical vaccine vector in man. Recently attempts have been made to overcome this problem by using an artificial adjuvant vector composed of the enzymatically active A1 fragment of CT (CTA1) linked to two Ig binding domains of staphylococcal protein A. The resulting CTA1-DD fusion protein binds B lymphocytes specifically, has no systemic toxicity and has similar adjuvant properties to CT holotoxin when given by parenteral routes (8)+. However, preliminary indications are that it may have only limited effects when given orally.
Whereas B lymphocytes play a central role in the adjuvant effects of CT and in particular, CTA1-DD (8, 13), the particulate nature of iscoms allows them to preferentially target and activate accessory cells such as macrophages and dendritic cells (DC) (14-17). Thus, the mucosal adjuvant effects of CT are dependent on the presence of IL4 dependent B cell follicles in Peyer's patches (PP), but not on IL12 (3, 13). In contrast, iscoms show an opposite pattern of requirements, inducing normal responses in IL4KO, but not in IL12KO mice (3, 17, and 18). In addition whereas CT may inhibit many functions of macrophages, iscoms stimulate the production of mediators such as IL1, IL6 and IL12 from macrophages and DC and in vivo depletion of macrophages markedly reduces the adjuvant effects of iscoms (15, 18-22).
Thus, iscoms and CTA1 and its derivatives use different anatomical routes and immune mechanisms to induce mucosal immune responses.